Vaginal suppository for treating group B Streptococcus infection

ABSTRACT

A composition for treatment of bacterial infections of the vagina is disclosed which comprises a lytic enzyme composition specific for the infecting bacteria, and a carrier for delivering said lytic enzyme. The lytic enzyme is which is specific for the group B  Streptococcus , is genectically coded for by a specific bacteriophage.

The following application is a continuation-in-part of Ser. No.09/497,495 filed Apr. 18, 2000 now U.S. Pat. No. 6,238,661, which is acontinuation of Ser. No. 09/395,636 filed Sep. 14, 1999) now U.S. Pat.No. 6,056,954 which is a continuation-in-part of U.S. patent applicationSer. No. 08/962,523, filed Oct. 31, 1997, now U.S. Pat. No. 5,997,862.

DESCRIPTION BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention discloses a method and composition for thetreatment of bacterial infections by the use of a lysing enzyme blendedwith an appropriate carrier suitable for the treatment of the infection.

2. Description of the Prior Art

In the past, antibiotics have been used to treat various infections. Thework of Selman Waksman in the introduction and production ofStreptomycetes, Dr. Fleming's discovery of penicillin, are well known aswell as the work of numerous others in the field of antibiotics. Overthe years, there have been additions and chemical modifications to the“basic” antibiotics in attempts to make them more powerful, or to treatpeople allergic to these antibiotics.

Others have found new uses for these antibiotics. U.S. Pat. No.5,260,292 (Robinson et al.) discloses the topical treatment of acne withaminopenicillins. The method and composition for topically treating acneand acneiform dermal disorders includes applying an amount of anantibiotic selected from the group consisting of ampicillin,amoxicillin, other aminopenicillins, and cephalosporins, and derivativesand analogs thereof, effective to treat the acne and acneiform dermaldisorders. U.S. Pat. No. 5,409,917 (Robinson et al.) discloses thetopical treatment of acne with cephalosporins.

However, as more antibiotics have been prescribed or used at an everincreasing rate for a variety of illnesses, increasing numbers ofbacteria have developed a resistance to antibiotics. Larger doses ofstronger antibiotics are now being used to treat ever more resistantstrains of bacteria. Multiple antibiotic resistant bacteria haveconsequently developed. The use of more antibiotics and the number ofbacteria showing resistance has led to increasing the amount of timethat the antibiotics need to be used. Broad, non-specific antibiotics,some of which have detrimental effects on the patient, are now beingused more frequently. Also, antibiotics do not easily penetrate mucuslinings. Additionally, the number of people allergic to antibioticsappears to be increasing.

Consequently, other efforts have been sought to first identify and thenkill bacteria.

Attempts have been made to treat bacterial diseases with by the use ofbacteriophages. U.S. Pat. No. 5,688,501 (Merril, et al.) discloses amethod for treating an infectious disease caused by bacteria in ananimal with lytic or non-lytic bacteriophages that are specific forparticular bacteria.

U.S. Pat. No. 4,957,686 (Norris) discloses a procedure of improveddental hygiene which comprises introducing into the mouth bacteriophagesparasitic to bacteria which possess the property of readily adhering tothe salivary pellicle.

It is to be noted that the direct introduction of bacteriophages into ananimal to prevent or fight diseases has certain drawbacks. Specifically,the bacteria must be in the right growth phase for the phage to attach.Both the bacteria and the and the phage have to be in the correct andsynchronized growth cycles. Additionally, there must be the right numberof phages to attach to the bacteria; if there are too many or too fewphages, there will either be no attachment or no production of thelysing enzyme. The phage must also be active enough. The phages are alsoinhibited by many things including bacterial debris from the organism itis going to attack. Further complicating the direct use of bacteriophageto treat bacterial infections is the possibility of immunologicalreactions, rendering the phage non-functional.

Consequently, others have explored the use of other safer and moreeffective means to treat and prevent bacterial infections.

U.S. Pat. No. 5,604,109 (Fischetti et al.) relates to the rapiddetection of Group A streptococci in clinical specimens, through theenzymatic digestion by a semi-purified Group C streptococcal phageassociated lysin enzyme. The lytic enzyme of this patent is used in U.S.Pat. No. 5,997,862(Fischetti, et. al.), U.S. Pat. No. 5,985,271,(Fischetti et al.) and U.S. Pat. No. 6,017,528 (Fischetti et al.) whichdisclose the use of an oral delivery mode, such as a candy, chewing gum,lozenge, troche, tablet, a powder, an aerosol, a liquid or a liquidspray, containing a lysin enzyme produced by group C streptococcalbacteria infected with a C1 bacteriophage for the prophylactic andtherapeutic treatment of Streptococcal A throat infections, commonlyknown as strep throat.

U.S. Pat. No. 6,056,955 (Fischetti et al.) discloses the topicaltreatment of streptococcal infections.

SUMMARY OF THE INVENTION

The method for obtaining and purifying the lytic enzyme produced by abacteria infected with the bacteriophage is known in the art. Somerecent evidence suggests that the phage enzyme that lyses thestreptococcus organism may actually be a bacterial enzyme that is usedto construct the cell wall and the phage. While replicating in thebacterium, a phage gene product may cause the upregulation orderepression of bacterial enzyme for the purpose of releasing thebacteriophage. These bacterial enzymes may be tightly regulated by thebacterial cell and are used by the bacteria for the construction andassembly of the cell wall.

The use of these lytic enzymes for the prophylactic and therapeutictreatment of bacterial diseases, however, has not been explored, exceptby the inventors of the present invention. Consequently, the presentinvention discloses the extraction and use of a variety of bacterialphage associated lytic enzymes for the treatment of a wide variety ofillnesses caused by bacterial infections.

The use of phage associated lytic enzymes produced by the infection of abacteria with a bacteria specific phage has numerous advantages for thetreatment of diseases. As the phage are targeted for specific bacteria,the lytic enzymes do not interfere with normal flora. Also, lytic phagesprimarily attack cell wall structures which are not affected by plasmidvariation. The actions of the lytic enzymes are fast and do not dependon bacterial growth.

Lytic enzymes can be directed to the mucosal lining, where, inresidence, they will be able to kill colonizing bacteria.

It is an object of the invention to use phage associated enzymes toprophylactically and therapeutically treat bacterial diseases.

The invention (which incorporates U.S. Pat. No. 5,604,109 in itsentirety by reference) uses an enzyme produced by the bacterial organismafter being infected with a particular bacteriophage as either aprophylactic treatment for preventing those who have been exposed toothers who have the symptoms of an infection from getting sick, or as atherapeutic treatment for those who have already become ill from theinfection. The present invention is based upon the discovery that phagelytic enzymes specific for bacteria infected with a specific phage caneffectively and efficiently break down the cell wall of the bacterium inquestion. At the same time, in most if not all cases, the semipurifiedenzyme is lacking in mammalian cell receptors and therefore isnon-destructive to mammalian proteins and tissues when present duringthe digestion of the bacterial cell wall. The same general techniqueused to produce and purify the lysin enzyme in U.S. Pat. No. 5,604,109may be used to manufacture other lytic enzymes produced by bacteriainfected with a bacteriophage specific for that bacteria. Depending onthe bacteria, there may be variations in the growth media andconditions.

In one embodiment of the invention, the prophylactic and therapeutictreatment of a variety of illnesses caused by Streptococcal pneumoniae,Streptococcus fasciae, and Hemophilus influenza are disclosed. Inanother embodiment of the invention, gram negative bacterial infectionscaused by Listeria, Salmonella, E. coli, and Campylobacter, are treatedby the use of lytic enzymes. These and other bacteria, which can infectthe digestive system, can be treated by incorporating the lytic enzymesin suppository enemas, in syrups, or in other carriers to get directlyto the site of the infection(s).

In another embodiment of the invention, lytic enzymes are incorporatedinto bandages to prevent or treat infections of burns and wounds. In yetanother embodiment of the invention, the lytic enzymes of phageassociated with Staphylococcus or Pseudomonas are incorporated intobandages to prevent or treat infections of burns and wounds.

Vaginal infections caused by Group B Streptococcus can cause prematurebirth and subsequent complications resulting in neonatal sepsis. Lysinincorporated into tampons specific for group B strep would preventinfection of the neonate during birth without disturbing normal vaginalflora so that women would not be overcome by yeast infection as a resultof antibiotic therapy.

In another embodiment of the invention, eye drops containing lyticenzymes of Hemophilus, Pseudomonas, and/or Staphylococcus can be used todirectly treat eye infections. Treatment with lytic enzymes are fasterand more expedient than with antibiotics.

In yet another embodiment of the invention the phage associated lyticenzyme is put into a carrier which is placed in an inhaler to treat orprevent the spread of diseases localized in the mucus lining of the oralcavity and lungs. Specific lytic enzymes for tuberculosis have beenisolated and can be used.

In another embodiment of the invention the lytic enzyme is administeredin the form of a candy, chewing gum, lozenge, troche, tablet, a powder,an aerosol, a liquid, a liquid spray, or toothpaste for the preventionor treatment of bacterial infections associated with upper respiratorytract illnesses.

In another embodiment of the invention, species specific lytic enzymescan be used in the treatment of bacterial infections associated withtopical or dermatological infections, administered in the form of atopical ointment or cream. In another embodiment of the invention, thelytic enzyme would be administered in an aqueous form. In yet anotherembodiment of the invention, lysostaphin, the enzyme which lysesStaphylococcus aureus, can be included in the therapeutic agent. In afurther embodiment of the invention, conventional antibiotics may beincluded in the therapeutic agent with the lytic enzyme, and with orwithout the presence of lysostaphin. More than one lytic enzyme may alsobe included in the prophylactic or therapeutic agent.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is an electron micrograph of group A streptococci treated withlysin showing the collapse of the cell wall and the cell contentspouring out.

DETAILED DESCRIPTION OF THE INVENTION

The method for treating bacterial infections comprises treating theinfection with a therapeutic agent comprising an effective amount of atleast one lytic enzyme produced by a bacteria infected with abacteriophage specific for the bacteria. The lytic enzyme is preferablyin an environment having a pH which allows for activity of said lyticenzyme.

The lytic enzyme can be used for the treatment or prevention ofHemophilus influenza, Pseudomonas, Streptococcus pneumoniae,Streptococcus fasciae, Streptococcus group B, Listeria, Salmonella, E.coli, Campylobacter, and other bacteria, and any combination thereof.

For example, if there is a bacterial infection of the upper respiratorytract, the infection can be prophylactically or therapeutically treatedwith a composition comprising an effective amount of at least one lyticenzyme produced by a bacteria being infected with a bacteriophagespecific for that bacteria, and a carrier for delivering the lyticenzyme to a mouth, throat, or nasal passage. It is preferred that thelytic enzyme is in an environment having a pH which allows for activityof the lytic enzyme. If an individual has been exposed to someone withthe upper respiratory disorder, the lytic enzyme will reside in themucosal lining and prevent any colonization of the infecting bacteria.

Two examples of bacteria which infect the upper respiratory system areStreptococcus pneumoniae and Hemophilus influenzae. In recent years,there has been an increase in the number of people, particularlychildren and the elderly, that are infected or are carriers ofpenicillin resistant Streptococcus pneumoniae and Hemophilus. Whilethese bacteria are normally harmless residents of the host, they areopportunistic organisms that are able to cause infections when theresistance of the host has been compromised. By eliminating or reducingthe number of these organisms in the upper respiratory tract, there willbe a commensurate reduction in the number of infections by thesebacteria.

Infection of the Hemophilus bacteria by Bacteriophage HP1 (a member ofthe P2-like phage family with strong similarities to coliphages P2 and186, and some similarity to the retronphage Ec67) produces a lyticenzyme capable of lysing the bacteria. The lytic enzyme forStreptococcus pneumoniae, previously identified as anN-acetyl-muramoyl-L-alanine amidase, is produced by the infectingStreptococcus pneumoniae with the Pal bacteriophage. The therapeuticagent can contain either or both of the lytic enzymes produced by thesetwo bacteria, and may contain other lytic enzymes for other bacteria.The composition which may be used for the prophylactic and therapeutictreatment of a strep infection includes the lysin enzyme and a means ofapplication (such as a carrier system or an oral delivery mode) to reachthe mucosal lining of the oral and nasal cavity, such that the enzyme isput in the carrier system or oral delivery mode to reach the mucosalining. Another infection which can be treated prophylactically isStreptococcus group A, which can produce what is commonly known as“strep” throat. When group C Streptococci are infected with a C1bacteriophage, a lysin enzyme is produced specific for the lysing ofStreptococcus group A.

Prior to, or at the time the lysin enzyme is put in the carrier systemor oral delivery mode, it is preferred that the enzyme be in astabilizing buffer environment for maintaining a pH range between about4.0 and about 9.0, more preferably between about 5.5 and about 7.5 andmost preferably at about 6.1.

The stabilizing buffer should allow for the optimum activity of thelysin enzyme. The buffer may be a reducing reagent, such asdithiothreitol. The stabilizing buffer may also be or include a metalchelating reagent, such as ethylenediaminetetracetic acid disodium salt,or it may also contain a phosphate or citrate-phosphate buffer.

Means of application include, but are not limited to direct, indirect,carrier and special means or any combination of means. Directapplication of the lytic enzyme may be by nasal sprays, nasal drops,nasal ointments, nasal washes, nasal injections, nasal packings,bronchial sprays and inhalers, or indirectly through use of throatlozenges, or through use of mouthwashes or gargles, or through the useof ointments applied to the nasal nares, the bridge of the nose, or theface or any combination of these and similar methods of application. Theforms in which the lysin enzyme may be administered include but are notlimited to lozenges, troches, candies, injectants, chewing gums,tablets, powders, sprays, liquids, ointments, and aerosols.

The lozenge, tablet, or gum into which the lytic enzyme is added maycontain sugar, corn syrup, a variety of dyes, non-sugar sweeteners,flavorings, any binders, or combinations thereof Similarly, any gumbased products may contain acacia, carnauba wax, citric acid, cornstarch, food colorings, flavorings, non-sugar sweeteners, gelatin,glucose, glycerin, gum base, shellac, sodium saccharin, sugar, water,white wax, cellulose, other binders, and combinations thereof.

Lozenges may further contain sucrose, corn starch, acacia, gumtragacanth, anethole, linseed, oleoresin, mineral oil, and cellulose,other binders, and combinations thereof In another embodiment of theinvention, sugar substitutes are used in place of dextrose, sucrose, orother sugars.

The enzyme may also be placed in a nasal spray, wherein the nasal sprayis the carrier. The nasal spray can be a long acting or timed releasespray, and can be manufactured by means well known in the art. Aninhalant may also be used, so that the phage enzyme may reach furtherdown into the bronchial tract, including into the lungs.

Any of the carriers for the lytic enzyme may be manufactured byconventional means. However, it is preferred that any mouthwash orsimilar type products not contain alcohol to prevent denaturing of theenzyme. Similarly, when the lytic enzyme is being placed in a coughdrop, gum, candy or lozenge during the manufacturing process, suchplacement should be made prior to the hardening of the lozenge or candybut after the cough drop or candy has cooled somewhat, to avoid heatdenaturation of the enzyme.

The enzyme may be added to these substances in a liquid form or in alyophilized state, whereupon it will be solubilized when it meets bodyfluids such as saliva. The enzyme may also be in a micelle or liposome.

The effective dosage rates or amounts of the lytic enzyme to treat theinfection will depend in part on whether the lytic will be usedtherapeutically or prophylactically, the duration of exposure of therecipient to the infectious bacteria, the size and weight of theindividual, etc. The duration for use of the composition containing theenzyme also depends on whether the use is for prophylactic purposes,wherein the use may be hourly, daily or weekly, for a short time period,or whether the use will be for therapeutic purposes wherein a moreintensive regimen of the use of the composition may be needed, such thatusage may last for hours, days or weeks, and/or on a daily basis, or attimed intervals during the day. Any dosage form employed should providefor a minimum number of units for a minimum amount of time. Theconcentration of the active units of enzyme believed to provide for aneffective amount or dosage of enzyme may be in the range of about 100units/ml to about 100,000 units/ml of fluid in the wet or dampenvironment of the nasal and oral passages, and possibly in the range ofabout 100 units/mi to about 10,000 units/mi. More specifically, timeexposure to the active enzyme units may influence the desiredconcentration of active enzyme units per ml. It should be noted thatcarriers that are classified as “long” or “slow” release carriers (suchas, for example, certain nasal sprays or lozenges) could possess orprovide a lower concentration of active (enzyme) units per ml, but overa longer period of time, whereas a “short” or “fast” release carrier(such as, for example, a gargle) could possess or provide a highconcentration of active (enzyme) units per ml, but over a shorter periodof time. The amount of active units per ml and the duration of time ofexposure depends on the nature of infection, whether treatment is to beprophylactic or therapeutic, and other variables.

While this treatment may be used in any mammalian species, the preferreduse of this product is for a human.

This composition and method may also be used for the treatment ofStreptococcus A infections of the respiratory tract. When using thiscomposition for a Streptococcus A infection, the lysin phage enzymeshould be used for the prophylactic prevention of Streptococcusinfections. Similarly, in another embodiment of the invention, thismethod may be used for the therapeutic and, preferably, the prophylactictreatment of tuberculosis. In a preferred embodiment of the invention,the phage associated lysing enzyme for Mycobacteria tuberculosis isplaced in a carrier in an inhaler. The carrier may be sterile water or awater base, or any other carrier used in an inhaler for dispersing drugsinto the bronchial tract. The phage associated lytic enzyme specific fortuberculosis is subject to the same conditions as the phage associatedlytic enzyme for other lytic enzymes. Specifically, prior to, or at thetime the enzyme is put in the carrier system or oral delivery mode, itis preferred that the enzyme be in a stabilizing buffer environment formaintaining a pH range between about 4.0 and about 9.0.

The stabilizing buffer should allow for the optimum activity of thelytic enzyme. The buffer may be a reducing reagent, such asdithiothreitol. The stabilizing buffer may also be or include a metalchelating reagent, such as ethylenediaminetetracetic acid disodium salt,or it may also contain a phosphate or citrate-phosphate buffer.

For the prophylactic and therapeutic treatment of tuberculosis, thephage associated lytic enzyme associated with tuberculosis may also beapplied by direct, indirect, carriers and special means or anycombination of means. Direct application of the lytic enzyme may be bynasal sprays, nasal drops, nasal ointments, nasal washes, nasalinjections, nasal packings, bronchial sprays and inhalers, or indirectlythrough use of throat lozenges, or through use of mouthwashes orgargles, or through the use of ointments applied to the nasal nares, thebridge of the nose, or the face or any combination of these and similarmethods of application. The forms in which the lytic enzyme may beadministered include but are not limited to lozenges, troches, candies,injectants, chewing gums, tablets, powders, sprays, liquids, ointments,and aerosols. For the therapeutic treatment of tuberculosis, thebronchial sprays and aerosols are most beneficial, as these carriers, ormeans of distributing the composition, allow the lytic enzyme to reachthe bronchial tubes and the lungs. An appropriate transport carrier maybe attached to the enzyme to transport the enzyme across the cellmembrane to the site of the bacteria.

The lozenge, tablet, or gum into which the lytic enzyme is added maycontain sugar, corn syrup, a variety of dyes, non-sugar sweeteners,flavorings, any binders, or combinations thereof Similarly, any gumbased products may contain acacia, carnauba wax, citric acid, cornstarch, food colorings, flavorings, non-sugar sweeteners, gelatin,glucose, glycerin, gum base, shellac, sodium saccharin, sugar, water,white wax, cellulose, other binders, and combinations thereof Lozengesmay further contain sucrose, corn starch, acacia, gum tragacanth,anethole, linseed, oleoresin, mineral oil, and cellulose, other binders,and combinations thereof In another embodiment of the invention, sugarsubstitutes are used in place of dextrose, sucrose, or other sugars.However, to tackle bacterial infections in the lung, the use of aninhaler carrier the lytic enzyme in a carrier is preferred.

Another use of a lytic enzyme is for the treatment of bacterialinfections of the digestive tract. The method for treating a bacterialinfection of the digestive tract comprises treating the bacterialinfection with composition comprising an effective amount of at leastone lytic enzyme produced by a bacteria infected with a bacteriophagespecific for the bacteria, and a carrier for delivering said lyticenzyme to the digestive tract. In a preferred embodiment of theinvention, the bacterial infections being treated are being caused bygram negative bacteria selected from the group consisting of Listeria,Salmonella, E. coli, and Campylobacter. However, this method andcomposition will effectively treat other bacteria, when the appropriatelytic enzyme is used.

In a preferred embodiment of the invention, the carrier is selected fromthe group consisting of suppository enemas, syrups, or enteric coatedpills. These proposed carriers can be made by conventional methods.However, the only difference in their manufacture is that the enzymebeing placed in the carrier must not be allowed to denature. To thatend, the enzyme should be incorporated into a carrier which does notcontain alcohol, and which has been cooled to a temperature that willnot cause the denaturing of the enzyme. The enzyme may be incorporatedin a lyophilized state, or maybe incorporated in a liposome before beingplaced in the suppository, syrup or enteric coated pill. The enzymeplaced in the composition or carrier should be in an environment havinga pH which allows for activity of the lytic enzyme. To this end, the pHof the composition is preferably kept in a range of between about 2 andabout 11, more preferably in a range of between about between about 4.0and about 9.0, and even more preferably at a pH range of between about5.5 and about 7.5. As described above with the other lytic enzyme, thepH can be moderated by the use of a buffer. The buffer may contain areducing agent, and more specifically dithiothreitol. The buffer mayalso be a metal chelating reagent, such as ethylenediaminetetraceticdisodium salt or the buffer may contain a citrate-phosphate buffer. Aswith all compositions described in this patent, the composition may,further include a bactericidal or bacteriostatic agent as apreservative.

The lytic enzyme is preferably present in a concentration of about 100to about 500,000 active enzyme units per milliliter of fluid in the wetenvironment of the gastrointestinal tract, preferably about 100 to about100,000 active enzyme units per milliliter of fluid, and preferablypresent in a concentration of about 100 to about 10,000 active enzymeunits per milliliter of fluid in the wet environment of thegastrointestinal tract.

The suppository is known in the art, and is made of glycerin, fattyacids, and similar type substances that dissolve at body temperature. Asthe suppository dissolves, the phage associated lytic enzyme will bereleased.

Another composition and use of the lytic enzyme is for the therapeuticor prophylactic treatment of bacterial infections of burns and wounds ofthe skin. The composition comprises an effective amount of at least onelytic enzyme produced by a bacteria infected with a bacteriophagespecific for the bacteria and a carrier for delivering at least onelytic enzyme to the wounded skin. The mode of application for the lyticenzyme includes a number of different types and combinations of carrierswhich include, but are not limited to an aqueous liquid, an alcohol baseliquid, a water soluble gel, a lotion, an ointment, a nonaqueous liquidbase, a mineral oil base, a blend of mineral oil and petrolatum,lanolin, liposomes, protein carriers such as serum albumin or gelatin,powdered cellulose carmel, and combinations thereof. A mode of deliveryof the carrier containing the therapeutic agent includes but is notlimited to a smear, spray, a time-release patch, a liquid absorbed wipe,and combinations thereof The lytic enzyme may be applied to a bandageeither directly or in one of the other carriers. The bandages may besold damp or dry, wherein the enzyme is in a lyophilized form on thebandage. This method of application is most effective for the treatmentof burns.

The carriers of the compositions of the present invention may comprisesemi-solid and gel-like vehicles that include a polymer thickener,water, preservatives, active surfactants or emulsifiers, antioxidants,sun screens, and a solvent or mixed solvent system. U.S. Pat. No.5,863,560 (Osborne) discusses a number of different carrier combinationswhich can aid in the exposure of the skin to a medicament.

Polymer thickeners that may be used include those known to one skilledin the art, such as hydrophilic and hydroalcoholic gelling agentsfrequently used in the cosmetic and pharmaceutical industries.Preferably, the hydrophilic or hydroalcoholic gelling agent comprises“CARBOPOL.RTM.” (B.F. Goodrich, Cleveland, Ohio), “HYPAN.RTM.” (KingstonTechnologies, Dayton, N.J.), “NATROSOL.RTM.” (Aqualon, Wilmington,Del.), “KLUCEL.RTM.” (Aqualon, Wilmington, Del.), or “STABILEZE.RTM.”(ISP Technologies, Wayne, N.J.). Preferably, the gelling agent comprisesbetween about 0.2% to about 4% by weight of the composition. Moreparticularly, the preferred compositional weight percent range for“CARBOPOL.RTM.” is between about 0.5% to about 2%, while the preferredweight percent range for “NATROSOL.RTM.” and “KLUCEL.RTM.” is betweenabout 0.5% to about 4%. The preferred compositional weight percent rangefor both “HYPAN.RTM.” and “STABILEZE.RTM.” is between about 0.5% toabout 4%.

“CARBOPOL.RTM.” is one of numerous cross-linked acrylic acid polymersthat are given the general adopted name carbomer. These polymersdissolve in water and form a clear or slightly hazy gel uponneutralization with a caustic material such as sodium hydroxide,potassium hydroxide, triethanolamine, or other amine bases.“KLUCEL.RTM.” is a cellulose polymer that is dispersed in water andforms a uniform gel upon complete hydration. Other preferred gellingpolymers include hydroxyethylcellulose, cellulose gum, MVE/MA decadienecrosspolymer, PVM/MA copolymer, or a combination thereof.

Preservatives may also be used in this invention and preferably compriseabout 0.05% to 0.5% by weight of the total composition. The use ofpreservatives assures that if the product is microbially contaminated,the formulation will prevent or diminish microorganism growth. Somepreservatives useful in this invention include methylparaben,propylparaben, butylparaben, chloroxylenol, sodium benzoate, DMDMHydantoin, 3-Iodo-2-Propylbutyl carbamate, potassium sorbate,chlorhexidine digluconate, or a combination thereof.

Titanium dioxide may be used as a sunscreen to serve as prophylaxisagainst photosensitization. Alternative sun screens include methylcinnamate. Moreover, BHA may be used as an antioxidant, as well as toprotect ethoxydiglycol and/or dapsone from discoloration due tooxidation. An alternate antioxidant is BHT.

Pharmaceuticals for use in all embodiments of the invention includeantimicrobial agents, anti-inflammatory agents, antiviral agents, localanesthetic agents, corticosteroids, destructive therapy agents,antifungals, and antiandrogens. In the treatment of acne, activepharmaceuticals that may be used include antimicrobial agents,especially those having anti-inflammatory properties such as dapsone,erythromycin, minocycline, tetracycline, clindamycin, and otherantimicrobials. The preferred weight percentages for the antimicrobialsare 0.5% to 10%.

Local anesthetics include tetracaine, tetracaine hydrochloride,lidocaine, lidocaine hydrochloride, dyclonine, dyclonine hydrochloride,dimethisoquin hydrochloride, dibucaine, dibucaine hydrochloride,butambenpicrate, and pramoxine hydrochloride. A preferred concentrationfor local anesthetics is about 0.025% to 5% by weight of the totalcomposition. Anesthetics such as benzocaine may also be used at apreferred concentration of about 2% to 25% by weight.

Corticosteroids that may be used include betamethasone dipropionate,fluocinolone actinide, betamethasone valerate, triamcinolone actinide,clobetasol propionate, desoximetasone, diflorasone diacetate,amcinonide, flurandrenolide, hydrocortisone valerate, hydrocortisonebutyrate, and desonide are recommended at concentrations of about 0.01%to 1.0% by weight. Preferred concentrations for corticosteroids such ashydrocortisone or methylprednisolone acetate are from about 0.2% toabout 5.0% by weight.

Destructive therapy agents such as salicylic acid or lactic acid mayalso be used. A concentration of about 2% to about 40% by weight ispreferred. Cantharidin is preferably utilized in a concentration ofabout 5% to about 30% by weight. Typical antifungals that may be used inthis invention and their preferred weight concentrations include:oxiconazole nitrate (0.1% to 5.0%), ciclopirox olamine (0.1% to 5.0%),ketoconazole (0.1% to 5.0%), miconazole nitrate (0.1% to 5.0%), andbutoconazole nitrate (0.1% to 5.0%). For the topical treatment ofseborrheic dermatitis, hirsutism, acne, and alopecia, the activepharmaceutical may include an antiandrogen such as flutamide orfinasteride in preferred weight percentages of about 0.5% to 10%.

Typically, treatments using a combination of drugs include antibioticsin combination with local anesthetics such as polymycin B sulfate andneomycin sulfate in combination with tetracaine for topical antibioticgels to provide prophylaxis against infection and relief of pain.Another example is the use of minoxidil in combination with acorticosteroid such as betamethasone diproprionate for the treatment ofalopecia ereata. The combination of an anti-inflammatory such ascortisone with an antifungal such as ketoconazole for the treatment oftinea infections is also an example.

In one embodiment, the invention comprises a dermatological compositionhaving about 0.5% to 10% carbomer and about 0.5% to 10% of apharmaceutical that exists in both a dissolved state and a microparticulate state. The dissolved pharmaceutical has the capacity tocross the stratum corneum, whereas the micro particulate pharmaceuticaldoes not. Addition of an amine base, potassium, hydroxide solution, orsodium hydroxide solution completes the formation of the gel. Moreparticularly, the pharmaceutical may include dapsone, an antimicrobialagent having anti-inflammatory properties. A preferred ratio of microparticulate to dissolved dapsone is five or less.

In another embodiment, the invention comprises about 1% carbomer, about80-90% water, about 10% ethoxydiglycol, about 0.2% methylparaben, about0.3% to 3.0% dapsone including both micro particulate dapsone anddissolved dapsone, and about 2% caustic material. More particularly, thecarbomer may include “CARBOPOL.RTM. 980” and the caustic material mayinclude sodium hydroxide solution.

In a preferred embodiment, the composition comprises dapsone andethoxydiglycol, which allows for an optimized ratio of micro particulatedrug to dissolved drug. This ratio determines the amount of drugdelivered, compared to the amount of drug retained in or above thestratum corneum to function in the supracorneum domain. The system ofdapsone and ethoxydiglycol may include purified water combined with“CARBOPOL.RTM.” gelling polymer, methylparaben, propylparaben, titaniumdioxide, BHA, and a caustic material to neutralize the “CARBOPOL.RTM.”

Any of the carriers for the lytic enzyme may be manufactured byconventional means. However, if alcohol is used in the carrier, theenzyme should be in a micelle, liposome, or a “reverse” liposome, toprevent denaturing of the enzyme. Similarly, when the lytic enzyme isbeing placed in the carrier, and the carrier is, or has been heated,such placement should be made after the carrier has cooled somewhat, toavoid heat denaturation of the enzyme. In a preferred embodiment of theinvention, the carrier is sterile.

The enzyme may be added to these substances in a liquid form or in alyophilized state, whereupon it will be solubilized when it meets aliquid body.

The effective dosage rates or amounts of the lytic enzyme to treat theinfection, and the duration of treatment will depend in part on theseriousness of the infection, the duration of exposure of the recipientto the infectious bacteria, the number of square centimeters of skin ortissue which are infected, the depth of the infection, the seriousnessof the infection, and a variety of a number of other variables. Thecomposition may be applied anywhere from once to several times a day,and may be applied for a short or long term period. The usage may lastfor days or weeks. Any dosage form employed should provide for a minimumnumber of units for a minimum amount of time. The concentration of theactive units of enzyme believed to provide for an effective amount ordosage of enzyme may be in the range of about 100 units/ml to about500,000 units/ml of composition, preferably in the range of about 1000units/ml to about 100,000 units/ml, and most preferably from about10,000 to 100,000 units/ml. The amount of active units per ml and theduration of time of exposure depends on the nature of infection, and theamount of contact the carrier allows the lytic enzyme to have. It is tobe remembered that the enzyme works best when in a fluid environment.Hence, effectiveness of the enzyme is in part related to the amount ofmoisture trapped by the carrier. In another preferred embodiment, a mildsurfactant in an amount effective to potentiate the therapeutic effectof the lytic enzyme. Suitable mild surfactants include, inter alia,esters of polyoxyethylene sorbitan and fatty acids (Tween series),octylphenoxy polyethoxy ethanol (Triton-X series),n-Octyl-.beta.-D-glucopyranoside, n-Octyl-.beta.-D-thioglucopyranoside,n-Decyl-.beta.-D-glucopyranoside, n-Dodecyl-.beta.-D-glucopyranoside,and biologically occurring surfactants, e.g., fatty acids, glycerides,monoglycerides, deoxycholate and esters of deoxycholate.

In order to accelerate treatment of the infection, the therapeutic agentmay further include at least one complementary agent which can alsopotentiate the bactericidal activity of the lytic enzyme. Thecomplementary agent can be penicillin, synthetic penicillins bacitracin,methicillin, cephalosporin, polymyxin, cefaclor. Cefadroxil, cefamandolenafate, cefazolin, cefixime, cefinetazole, cefonioid, cefoperazone,ceforanide, cefotanme, cefotaxime, cefotetan, cefoxitin, cefpodoximeproxetil, ceftazidime, ceftizoxime, ceftriaxone, cefriaxone moxalactam,cefuroxime, cephalexin, cephalosporin C, cephalosporin C sodium salt,cephalothin, cephalothin sodium salt, cephapirin, cephradine,cefuroximeaxetil, dihydratecephalothin, moxalactam, loracarbef mafate,chelating agents and any combinations thereof in amounts which areeffective to synergistically enhance the therapeutic effect of the lyticenzyme.

Additionally, the therapeutic agent may further comprise the enzymelysostaphin for the treatment of any Staphylococcus aureus bacteria.Mucolytic peptides, such as lysostaphin, have been suggested to beefficacious in the treatment of S. aureus infections of humans(Schaffner et al., Yale J. Biol. & Med., 39:230 (1967) and bovinemastitis caused by S. aureus (Sears et al., J. Dairy Science, 71 (Suppl.1): 244(1988)). Lysostaphin, agene product of Staphylococcus simulans,exerts a bacteriostatic and bactericidal effect upon S. aureus byenzymatically degrading the polyglycine crosslinks of the cell wall(Browder et al., Res. Comm., 19: 393-400 (1965)). U.S. Pat. No.3,278,378 describes fermentation methods for producing lysostaphin fromculture media of S. staphylolyticus, later renamed S. simulans. Othermethods for producing lysostaphin are further described in U.S. Pat.Nos. 3,398,056 and 3,594,284. The gene for lysostaphin has subsequentlybeen cloned and sequenced (Recsei et al., Proc. Natl. Acad. Sci. USA,84: 1127-1131 (1987)). The recombinant mucolytic bactericidal protein,such as r-lysostaphin, can potentially circumvent problems associatedwith current antibiotic therapy because of its targeted specificity, lowtoxicity and possible reduction of biologically active residues.Furthermore, lysostaphin is also active against non-dividing cells,while most antibiotics require actively dividing cells to mediate theireffects (Dixon et al., Yale J. Biology and Medicine, 41: 62-68 (1968)).Lysostaphin, in combination with the lysin enzyme, can be used in thepresence or absence of the listed antibiotics. There is a degree ofadded importance in using both lysostaphin and the lysin enzyme in thesame therapeutic agent. Frequently, when a body has a bacterialinfection, the infection by one genus of bacteria weakens the body orchanges the bacterial flora of the body, allowing other potentiallypathogenic bacteria to infect the body. One of the bacteria thatsometimes co-infects a body is Staphylococcus aureus. Many strains ofStaphylococcus aureus produce penicillinase, such that Staphylococcus,Streptococcus, and other gram positive bacterial strains will not bekilled by standard antibiotics. Consequently, the use of the lysin andlysostaphin, possibly in combination with antibiotics, can serve as themost rapid and effective treatment of bacterial infections. In yetanother preferred embodiment, the invention may include mutanolysin, andlysozyme.

In preferred embodiments of the invention, the lytic enzymes forPseudomonas, Staphylococcus, and Streptococcus, jointly or individually,may be incorporated into the carrier, or into a bandage to be used onburn patients, or in a solution or cream carrier.

Yet another use of lytic enzymes is for the prophylactic or therapeutictreatment of vaginal infections. This treatment comprises treating thevaginal infection with an effective amount of at least one lytic enzymeproduced by a bacteria being infected with a bacteriophage specific forthat bacteria, wherein that lytic enzyme is incorporated in a carrier tobe placed in a vagina. The preferred carrier is a tampon, or vaginaldouche. A pad may also be used as a carrier, although it is not aseffective. While any number of bacteria could be treated using thiscomposition and method, it is believed that the most optimum use of thistreatment composition and method would be for the treatment of an E.coli and Streptococcus B infection. Vaginal infections caused by Group BStreptococcus can cause neonatal meningitis resulting in brain damageand premature death. Lytic enzyme incorporated into tampon specific forgroup B Strep would eliminate the group B organisms without disturbingnormal flora so that woman would not be overcome by yeast infection postantibiotic therapy. The use of the lytic enzyme in the vagina would bestprovide a prophylactic effect, although therapeutic use would also beadvisable.

To produce a pad or tampon containing the enzyme, the lytic enzyme canbe applied in a solution to the tampon, and allowed to dry. The lyticenzyme may be incorporated into the pad or tampon by any other meansknown in the art, including lyophilization, spraying, etc. The tamponsand pads may also be kept slightly moist, and in a sealed wrapper untilready for use. In that case, bactericide and bacteriostatic compoundsand inhibitors should be present in the tampons and pads. The method tobe used for incorporating the lytic enzyme into the tampon or pad can beone of the methods known in the art for incorporating a pharmaceuticalproduct. In another embodiment of the invention, the lytic enzyme isincorporated into a vaginal suppository. The vaginal suppository intowhich the lytic enzyme is being incorporated may be a standard vaginalsuppository, comprised of glyceride, alginate, starch, other standardbinders and any combinations thereof When using a tampon as the carrier,it is best to insert the tampon in the vagina and leave it in for up to12 hours to distribute the enzyme vaginally.

As with other lytic enzymes, it is preferable that the pH be kept in arange of about 4.0 and about 9.0 even more preferably at a pH range ofbetween about 5.5 and about 7.5. As described above with the other lyticenzyme, the pH can be moderated by the use of a buffer. The buffer maycontain a reducing agent, and more specifically dithiothreitol. Thebuffer may also contain a metal chelating reagent, such asethylenediaminetetracetic disodium salt or the buffer may be acitrate-phosphate buffer. As with all compositions described in thispatent, the composition may, further include a bactericidal orbacteriostatic agent as a preservative.

The lytic enzyme is preferably present in a concentration of about 100to about 500,000 active enzyme units per milliliter of fluid in the wetenvironment of the vaginal tract, preferably about 100 to about 100,000active enzyme units per milliliter of fluid, and preferably present in aconcentration of about 100 to about 10,000 active enzyme units permilliliter of fluid in the wet environment of the vaginal tract.

Another use of the invention is for the prophylactic and therapeutictreatment of eye infections. The method of treatment comprisesadministering eye drops which comprise an effective amount of at leastone lytic enzyme produced by the bacteria being infected with abacteriophage specific for the bacteria and a carrier capable of beingsafely applied to an eye, with the carrier containing the lytic enzyme.In a preferred embodiment of the invention, the bacteria being treatedis Hemophilus or Staphylococcus. The eye drops are in the form of anisotonic solution. The pH of the solution should be adjusted so thatthere is no irritation of the eye, which in turn would lead to possiblyinfection by other organisms, and possibly to damage to the eye. Whilethe pH range should be in the same range as for other lytic enzymes, themost optimal pH will be in the range of from 6.0 to 7.5. Similarly,buffers of the sort described above for the other lytic enzymes shouldalso be used. Other antibiotics which are suitable for use in eye dropsmay be added to the composition containing the lytic enzymes.Bactericides and bacteriostatic compounds may also be added.

It is to be remembered that all of the enzymes can be used forprophylactic and therapeutic treatments of the bacteria for which theenzymes are specific.

It is also to be remembered that a carrier may have more than one lyticenzyme. For instance, A throat lozenge may comprise just a lysin enzyme(which lyses the Streptococcus A strain causing “strep” throat, or itmay also include the lytic enzymes for Hemophilus. Similarly, thecarrier for treating burns and wounds, or infections of the skin, maycontain just one lytic enzyme, or a combination of lytic enzymes, forthe treatment of Pseudomonas, Streptococcus, Staphylococcus, or anyother of a number of bacteria.

Lytic enzymes can also be used to fight dental caries. Specifically, alytic enzyme specific for Streptococcus mutans may be incorporated in atoothpaste or oral wash. Similarly, this lytic enzyme may also beincorporated into a chewing gum or lozenge. Any other carrier can beused that allows for the exposure of the mouth, gums, and teeth to thelytic enzyme.

The lytic enzyme may also be incorporated in a lyophilized or dried formin tooth powder. If the lytic enzyme is to be used in an oral wash, itis preferred that the oral wash not contain any alcohol, so as to notdenature the enzyme. The enzyme can also be in a liposome when mixed inwith the toothpaste or oral wash. The concentrations of the enzyme unitsper ml of toothpaste or mouth wash can be in the range of from about 100units/ml to about 500,000 units/ml of composition, preferably in therange of about 1000 units/ml to about 100,000 units/ml, and mostpreferably from about 10,000 to 100,000 units/ml. The pH of thetoothpaste or oral wash should be in a range that allows for the optimumperformance of the enzyme, while not causing any discomfort to the userof the toothpaste or oral wash.

The following example illustrates the isolation of the lytic enzyme.

Phage Associated Enzyme

The group C phage lysin enzyme is prepared as follows:

Group C streptococcal strain 26RP66 (ATCC #21597) or any other group Cstreptococcal strain is grown in Todd Hewitt medium at 37.degree. C. toan OD of 0.23 at 650 nm in an 18 mm tube. Group C bacteriophage (C1)(ATCC #21597-B1) at a titer of 5.times. 10.sup.6 is added at a ratio of1 part phage to 4 parts cells. The mixture is allowed to remain at37.degree. C. for 18 min at which time the infected cells are pouredover ice cubes to reduce the temperature of the solution to below15.degree. C. The infected cells are then harvested in a refrigeratedcentrifuge and suspended in 1/300th of the original volume in 0.1Mphosphate buffer, pH 6.1 containing 5.times. 10.sup.-3 M dithiothreitoland 10 ug of DNAase. The cells will lyse releasing phage and the lysinenzyme. After centrifugation at 100,000.times. g for 5 hrs to removemost of the cell debris and phage, the enzyme solution is aliquoted andtested for its ability to lyse Group A Streptococci.

The number of units/ml in a lot of enzyme is determined to be thereciprocal of the highest dilution of enzyme required to reduce theOD650 of a suspension of group A streptococci at an OD of 0.3 to 0.15 in15 minutes. In a typical preparation of enzyme 4.times.10.sup.5 to4.times.10.sup.6 units are produced in a single 12 liter batch.

The enzyme may be diluted in a stabilizing buffer containing theappropriate conditions for stability, maximum enzymatic activity,inhibitors of nonspecific reactions, and in some configurations containsspecific antibodies to the Group A carbohydrate. The preferredembodiment is to use a lyophilized reagent which can be reconstitutedwith water. The stabilizing buffer can comprise a reducing reagent,which can be dithiothreitol in a concentration from 0.001M to 1.0M,preferably 0.005M. The stabilizing buffer can comprise an immunoglobulinor immunoglobulin fragments in a concentration of 0.001 percent to 10percent, preferably 0.1 percent. The stabilizing buffer can comprise acitrate-phosphate buffer in a concentration from 0.001M to 1.0M,preferably 0.05M. The stabilizing buffer can have a pH value in therange from 5.0 to 9.0. The stabilizing buffer can comprise abactericidal or bacteriostatic reagent as a preservative. Suchpreservative can be sodium azide in a concentration from 0.001 percentto 0.1 percent, preferably 0.02 percent.

The preparation of phage stocks for lysin production is the sameprocedure described above for the infection of phage and group Cstreptococcus in the preparation of the lysin enzyme. However, insteadof pouring the infected cells over ice, the incubation at 37.degree. C.is continued for a total of 1 hour to allow lysis and release of thephage and also enzyme in the total volume. In order for the phage to beused for subsequent lysin production the residual enzyme must beinactivated or removed to prevent lysis from without of the group Ccells rather than phage infection.

The thin section electron micrograph of FIG. 1 shows the results of agroup A streptococci 1 treated for 15 seconds with lysin. The micrograph(25,000×magnification) shows the cell contents 2 pouring out through ahole 3 created in the cell wall 4 by the lysin enzyme.

This general technique can be used to produce other lytic enzymes forthe treatment of various bacterial infections.

Many modifications and variations of the present invention are possiblein light of the above teachings. It is, therefore, to be understoodwithin the scope of the appended claims the invention may be protectedotherwise than as specifically described.

What we claim is:
 1. A vaginal suppository for treating a Group BStreptococcus infection of the vagina comprising: (i) an effectiveamount of a lytic enzyme genetically coded for by a bacteriophagespecific for said Group B Streptococcus, said lytic enzyme beingspecific for said Group B Streptococcus whereby said lytic enzyme hasthe ability to digest a cell wall of said Group B Streptococcus; and(ii) a carrier for delivering said lytic enzyme to the vagina.